PROJECT SUMMARY Mechanosensory hair cell loss in the inner ear is a leading cause of hearing and balance disorders. Unfortunately hair cell loss is permanent as humans have little or no capacity for regenerative recovery of hair cells. Although there has been recent progress in the restoration of hair cells in mammals by promoting expression of genes and pathways used during development, functional restoration is largely incomplete and within a limited window of postnatal development. Potential new targets for promoting regeneration, however, are limited. We propose to undertake a high-throughput screen for transcriptional regulators of hair cell development and regeneration in the zebrafish lateral line, a mechanosensory system that is readily accessible to visualization and manipulation in living animals. While there are certainly differences in the organization of the lateral line and inner ear, there is fundamental conservation of many of the genes used in hair cell development and function. The CRISPR system uses easily synthesized guide RNAs to target specific genes and direct Cas9 nuclease to make mutagenic breaks. It is highly efficient, allowing modification of both gene alleles within the same cell and resulting in genetic loss of function. We demonstrate that we can use CRISPR to target expression of genes in the lateral line. We propose a systematic screen for rapid identification of genes that regulate hair cell development and regeneration.